CN102884174B - The screening technique of intestinal immune inhibitor - Google Patents
The screening technique of intestinal immune inhibitor Download PDFInfo
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- CN102884174B CN102884174B CN201080065776.1A CN201080065776A CN102884174B CN 102884174 B CN102884174 B CN 102884174B CN 201080065776 A CN201080065776 A CN 201080065776A CN 102884174 B CN102884174 B CN 102884174B
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Abstract
The present invention provide a kind of can the biological method of the immunity of the Effective selection struvite immunization that can effectively suppress enteral to induce.The present invention relates to suppress the screening technique of the inhibitor of struvite immunization in intestinal epithelial cell, the bacterial strain separated by this screening technique, compositions containing this bacterial strain.
Description
Technical field
The present invention relates to suppress the screening technique of immunity biological (Immunobiotics) of struvite immunization, the bacterial strain screened by the method, the new strains with described inhibitory action at enteral and there is the compositions of described bacterial strain.
Background technology
Discovery and the use of antibiotics (Antibiotics) are extremely important for medical development now.But, in recent years, the side effect of antibiotics or because sudden change occurs that the problems such as multi-drug resistant become discussion topic.Accordingly, with respect to " antibiotics ", " probiotic bacteria (probiotics) " this idea is the most well-known.Probiotic bacteria, refer to " bacterial flora in digestive tract, the beneficial microbe bringing beneficial effect for host and proliferating and promoting substances thereof can be improved ", recently germinate and to use these probiotic actions flora (bacterial flora) in (in oral cavity or enteral) in digestive tract, carry out preventing, improving this idea of disease while attempting to make flora health.
In mammals digestive tract, foreign bodies is being taken in internal intestinal, immune system maximum in body.Thus, regulating intestinal canal immunity, extremely important for mammiferous health.In recent years, in " probiotic bacteria ", as having the material of intestinal tract immune system regulatory function, occurring in that " immunity is biological " this concept, its application attracts tremendous attention.
Toll-like receptor (TLR) is present in panimmunity class cell, is well known because of the special molecular pattern of the pathogen such as its recognizable antibacterial, fungus, parasite and virus.TLR family is made up of the 11 kinds of protein being referred to as TLR1 to TLR11, it is believed that its most bacillary modulin of respective identification molecule (modulin) is different.It is believed that immunity biology regulates immunity by acting on the TLR expressed in intestinal tract immune system.
Recently, people begin one's study, and these immunity are biological can produce to intestinal tract immune system for what kind of impact, attempts the screening suitable bacterial strain as immunity biology.Such as patent document 1 discloses that and identified specifically from the oligonucleotide of Lactobacillus gasseri (Lactobacillusgasseri bacterium) by TLR9, lymphocyte blastogenesis demonstrates activity.In non-patent literature 1, disclose by stimulating the conversion cell expressing pig TLR2 with lactic acid bacteria, study the cytokine-expressing in this cell, thus evaluate the method as immune biological lactic acid bacteria.In non-patent literature 2, disclosing in the case of people's Caco2 cell and escherichia coli k88 strain being co-cultured, if co-cultured with specific lactic acid bacteria further, the most struvite response is suppressed.In non-patent literature 3, disclose in swine intestinal epithelium cells, biological because of the immunity of enterotoxigenic E．Coli (EnterotoxigenicE.coli) induction inflammation about suppression, according to the suppression producing IL-6 or IL-8, the immunity filtering out performance anti-inflammatory is biological.
Prior art literature
Patent documentation
Patent documentation 1: Japanese Unexamined Patent Publication 2006-232790
Non-patent literature
Non-patent literature 1:Tohno etc., animal science magazine (2007) 78,195-205(Tohnoetal., AnimalScienceJournal (2007) 78,195-205)
Non-patent literature 2:Roselli etc., Britain's threpsology's magazine (2006), 95(Rosellietal., BritishJournalofNutrition (2006), 95)
Non-patent literature 3: Shimadzu etc. " the biological antiinflammatory action in intestinal epithelial cell of immunity " Japan's animal science can the 110th conference speech theme X27-05
Summary of the invention
Invention to solve the technical problem that
Inventors etc. study discovery, in the intestinal epithelial cell research immunity biochron using cattle, if using IL-6, IL-8, MCP-1, then effective evaluation can have the immune biological of struvite inhibitive ability of immunity.And, further research finds, uses intestinal epithelial cell, be used for evaluating by least two selected from IL-6, IL-8 and MCP-1, it is successfully separated the immunity biology with good struvite inhibitive ability of immunity with this evaluation methodology, uses this bacterial strain to be found that its mechanism.And by further investigation further, result confirms if made in the compositions being orally ingested containing this bacterial strain, then can improve the immunocompetence of individuality, thus complete the present invention.
Solve the technological means of technical problem
I.e., the present invention relates to Lactobacillus (Lactobacillus Pseudomonas) bacterium, itself and intestinal epithelial cell are co-cultured more than 2 days, thus the expression that the two or more struvite immunity-associated cell factor suppressed in the intestinal epithelial cell being exposed in pro-inflammatory cytokine in IL-6, IL-8 and MCP-1 is compared with the above-mentioned struvite immunity-associated cell factor in the intestinal epithelial cell not co-cultured.
Additionally, the described Lactobacillus bacterium of the present invention, the expression of suppression IL-6, IL-8 and MCP-1 these three.
And then, the described Lactobacillus of the present invention, more than the 30% of the expression of inflammatory immune relevant cell factor becomes anti-inflammatory.
Additionally, the described Lactobacillus of the present invention is lactobacillus casei (Lactobacilluscasei), sieve Yi Shi lactobacillus (Lactobacillusreuteri), lactobacillus rhamnosus (Lactobacillusrhamnosus), Lactobacillus salivarius (Lactobacillussalivarius), Lactobacillus Jensenii (Lactobacillusjensenii) or Lactobacillus gasseri (Lactobacillusgasseri).
The invention still further relates to lactobacillus casei (Lactobacilluscasei) OLL2768 bacterial strain (preserving number: FERMBP-11244).
Moreover, it relates to Lactobacillus Jensenii (Lactobacillusjensenii) TL2937 bacterial strain (preserving number: FERMBP-11272).
And then, the invention still further relates to a kind of suppress the screening technique of the inhibitor of struvite immunization in intestinal epithelial cell, it includes following operation:
(1) operation of the cell expressing Toll-like receptor (TLR) 2 and 4 is stimulated with candidate inhibitor；
(2) operation of the most post-stimulatory cell is stimulated with pro-inflammatory cytokine；
(3) operation of the expression of one or more the struvite immunity-associated cell factor in the cell after (2) moderate stimulation is measured；
Wherein, the expression of one or more the described struvite immunity-associated cell factor, compared with the comparison not carrying out described (1) operation, in the case of becoming anti-inflammatory, described candidate inhibitor has struvite immunization inhibition.
Further, the described method of the present invention, between (1) and (2), still further comprise the operation of the cell after (1 ') cleaning (1) moderate stimulation.
It addition, the described method of the present invention, when the struvite immunity-associated cell factor expression compared with the control, in the case of more than 30% becomes anti-inflammatory, regard as candidate inhibitor and there is struvite immunization inhibition.
It addition, the described method of the present invention, TLR2 and 4 comes from cattle, pig or people.
It addition, the described method of the present invention, pro-inflammatory cytokine is enterotoxigenic E．Coli (ETEC).
It addition, the described method of the present invention, the cell expressing TLR2 and 4 is intestinal epithelial cell.
It addition, the described method of the present invention, the struvite immunity-associated cell factor measuring expression is IL-6, IL-8 and MCP-1.
It addition, the described method of the present invention, struvite immunization inhibitor is the biological or derivatives thereof (immunogenicity microorganism (immunogenics)) of immunity.
It addition, the invention still further relates to by the isolated immunity of described screening technique biological.
It addition, the described immunity biology of the present invention is Lactobacillus bacterium or Bifidobacterium bacterium.
The invention still further relates to a kind of inflammation inhibitor compositions, it comprises at least one in described bacterium or immunity biology.
It addition, the described inflammation inhibitor compositions of the present invention, it is used for preventing and/or dispose enteral inflammatory disease.
It addition, the invention still further relates to a kind of food compositions, it comprises described inflammation inhibitor compositions.
It addition, the invention still further relates to a kind of fodder compound, it comprises described inflammation inhibitor compositions.
Invention effect
By the screening technique of the present invention, it is possible to the immunity that Effective selection goes out the struvite immunization that can effectively suppress enteral to induce is biological.Further, the immunity gone out by the screening technique screening and separating of the present invention is biological, and once by immunocyte identification, then the mrna expression on immunocyte is composed and produced impact, even if in the presence of without this immunity biology, also can suppress struvite immunization.
Accompanying drawing explanation
Fig. 1 is to represent with the accompanying drawing of each expression for IL-6, IL-8 and MCP-1 in the BIE cell of examination lactic acid bacteria stimulation.With Student ' st inspection resolve relative to ETEC comparison 2 groups between statistical significant difference.As suppressing the expression of IL-6 and IL-8 that expression is the highest in each cytokine, and then also can suppress the bacterial strain of the expression of MCP-1, filter out OLL2768.
Fig. 2 is the chart representing the expression with each cytokines mRNA in OLL2768 bacterial strain post-stimulatory BIE cell.Transverse axis represents the time stimulated after starting, the longitudinal axis represent with non-stimulated be expressed as 1 time relative intensity.
Fig. 3 is to represent that OLL2768 bacterial strain stimulated after 48 hours, gives ETEC further when stimulating, the chart of the change of the expression of each cytokines mRNA in BIE cell.Transverse axis represent ETEC stimulate after elapsed time, the longitudinal axis represent with non-stimulated be expressed as 1 time relative intensity.
Fig. 4 a represents that BIE cell stimulates with ETEC after inoculating 3, measures the chart of the mrna expression amount of 12 kinds of cytokines after stimulating 12 hours.The longitudinal axis represent with non-stimulated be expressed as 1 time relative intensity.Statistical significant difference between resolving relative to 2 groups compareed with Student ' st inspection.As a result, the alternate cell selecting predictors as strong induction has gone out IL-1 α, IL-6, IL-8 and MCP-1.Fig. 4 b) represent that BIE cell stimulates with ETEC after inoculating 5, measure the chart of the mrna expression amount of the above-mentioned 4 kinds of alternate cell factors after stimulating 12 hours.Statistical significant difference between resolving relative to 2 groups compareed with Student ' st inspection.As a result, IL-6, IL-8 and MCP-1 have been filtered out as the cytokine should measured in an embodiment.
Fig. 5 is to represent the pre-stimulation carried out swine intestinal epithelium cells with the biological candidate bacterial strain of immunity 0,12,24 and 48 hours, the chart of the expression of the IFN-β after then stimulating 12 hours with polyinosinic acid (polyI:C).
Fig. 6 is to represent the pre-stimulation carried out swine intestinal epithelium cells with the biological candidate bacterial strain of immunity 0,12,24 and 48 hours, the chart of the expression of the MCP-1 after then stimulating 12 hours with polyinosinic acid.
Fig. 7 is to represent in each feedstuff district, the accompanying drawing of the testing result of the pathogenic Escherichia coli in feces.Upper figure represents that k99, figure below represent k88, and the black vaginal discharge on position shown in arrow represents and detected each antigen.Even if adding antibacterial as shown in the figure the most all can not prevent the infection of the enterotoxigenic E．Coli in 13 ~ 15 week old.
Fig. 8 is to represent in each feedstuff district, the chart of the concentration of the c reactive protein (CRP) in blood plasma.It is known that the CRP concentration in blood plasma can be as the index of internal inflammatory reaction, concentration height represents the internal reaction that is inflamed.
Fig. 9 is to represent that in each feedstuff district, granulocyte number is relative to the chart of the ratio of lymphocyte number.Granulocyte ratio is the highest, represents and the most easily activates natural immunity.
Figure 10 is to represent in each feedstuff district, the accompanying drawing of the testing result of the pathogenic Escherichia coli ETEC987 in feces.3rd district (without antibacterial, putting into TL2937 bacterial strain) are not even if detect ETEC987 in 13 ~ 15 week old yet.
Figure 11 is to represent in each feedstuff district, the chart of CRP concentration during 13 ~ 15 week old.In 3rd district, compared with other districts, CRP concentration is the lowest.
Figure 12 is to represent in each feedstuff district, the chart of complement the 2nd pathway activity.Identical with CRP concentration, can confirm that complement the 2nd pathway activity that significance is high in 3rd district.
Figure 13 is to represent in each feedstuff district, the chart of the body weight of 22 week old objects.
Figure 14 is the chart representing carcass weight when delivering for sale.
Detailed description of the invention
The present invention is a kind of to suppress the screening technique of the inhibitor of struvite immunization in intestinal epithelial cell, and it includes following operation:
(1) operation of the cell expressing Toll-like receptor (TLR) 2 is stimulated with candidate inhibitor；
(2) operation of the most post-stimulatory cell is stimulated with pro-inflammatory cytokine；
(3) operation of the expression of one or more the struvite immunity-associated cell factor in measurement cell after (2) moderate stimulation；
Wherein, the expression of the 1 or of more than two kinds described struvite immunity-associated cell factor, compared with the comparison not carrying out described (1) operation, in the case of being changed to anti-inflammatory, described candidate inhibitor has struvite immunization inhibition.
In the present invention, " struvite immunization inhibition " is the character representing and suppressing struvite immunization, and " struvite immunization inhibitor " is the preparation representing and having struvite immunization inhibition.Struvite immunization inhibitor includes the compounds such as microorganism, polypeptide such as such as immunity biology, but is not limited to this.In the present invention, " immunity is biological " refers to the probiotic bacteria in probiotic bacteria with immunoloregulation function.
In the present invention, " being changed to anti-inflammatory " is to represent the expression stimulating the struvite immunity-associated cell factor expressed by pro-inflammatory cytokine, exceedes the scope of statistical error to the change of suppression inflammatory reaction direction.Thus, such as if promoting the cytokine of inflammatory reaction, to the direction change making its expression reduce, if the cytokine of suppression inflammatory reaction, then to the direction change making its expression increase, or express the enhanced direction change expressed and reduce to suppression is momentary, but be not limited to this.Statistics parsing can be widely used all statistics analytic methods well known to the skilled person, can enumerate such as Student ' st inspection etc., but be not limited to this.During screening, in one or more the struvite immunity-associated cell factor, in the case of expression is changed to anti-inflammatory, it can be determined that candidate agent is struvite immunization inhibitor.Preferably in the two or more struvite immunity-associated cell factors, expression is changed to the situation of anti-inflammatory, the situation of more preferably more than 3 kinds.The expression of the whole struvite immunity-associated cell factor more preferably measured all is changed to the situation of anti-inflammatory.
In an embodiment of the invention, in the case of more than 30% when expression is changed to anti-inflammatory, it is judged that candidate inhibitor has struvite immunization inhibition.
In the present invention, the struvite immunity-associated cell factor, comprise the whole cytokine relevant to inflammatory reaction.The struvite immunity-associated cell factor, it is possible to distinguish the cytokine that there is the cytokine promoting inflammatory reaction character with there is suppression inflammatory reaction character.As having the cytokine promoting inflammatory reaction character, such as the inflammatory cytokine etc. such as IL-1 α, IL-1 β, IL-6, IL-7, IL-8, IL-12, IL-13, IL-17, IL-18, IFN-γ, MCP-1, TNF-α, LIF can be enumerated, but be not limited to this.As having the cytokine suppressing inflammatory reaction character, such as the anti-inflammatory cytokines such as IL-4, IL-10, TGF-β, the antiviral property cytokine such as IFN-α, IFN-β etc. can be enumerated, but be not limited to this.
It is known that Toll-like receptor (Tolllikereceptor:TLR) is a kind of pattern recognition receptors, activated by it, induce the immunne response signal cascade to pathogen and amplify.Additionally, it is well known that TLR family is highly present in vertebrates.In TLR family, TLR2 is to be the TLR being identified as part by the glycoprotein etc. of lipopolysaccharide, virus using lipoprotein, the Peptidoglycan of gram positive bacteria, lipoteichoic acid, the polysaccharide of fungus, the TLR, TLR4 that are identified as part such as glycoprotein of virus.Wherein, it is believed that enterotoxigenic E．Coli induces inflammation by TLR4, virus induces inflammation by TLR3, but immunity is biological regulates inflammatory response by TLR2.
Express the cell of TLR2, can be any cell, do not limit, including separating cell, cultivation cell, converting cell.TLR2 can be i.e. naturally to express, it is also possible to passes through cell transfecting so that it is forced expression.It is as noted previously, as TLR family to be highly present in vertebrates, therefore can use any animal, but preferably there is the animal of intestinal bacteria, particularly preferred mammal.In mammal, including the Rodents such as rat, mice, rabbit, cattle, horse, pig, sheep, monkey, goat, people etc., but it is not limited to this.
Express the cell of TLR2, the most also express one or more other TLR.As other TLR, such as TLR1, TLR3, TLR4, TLR5, TLR6, TLR9 etc. can be enumerated, but be not limited to this.As the TLR relevant to viral inflammation, such as TLR3, TLR9, preferably TLR3 can be enumerated.As the TLR relevant to bacterial inflammation, such as TLR1, TLR4, TLR5, TLR6, TLR9 etc., preferably TLR4 can be enumerated.
In the present invention, " intestinal epithelial cell " is present in the cell on vertebrates bowel lumen face, refers to the cell of the related immune responses such as the alimentation in participation intestinal or biophylaxis.It is known that in intestinal epithelial cell, the pattern recognition receptors headed by TLR is expressed, thus comprises the response of the signal pathway via TLR in the immunne response in intestinal.As it has been described above, TLR family is highly present in vertebrates, can be arbitrary vertebrates as vertebrates, but preferably there is the animal of intestinal bacteria, particularly preferred mammal.
The screening technique of the present invention, the first-selected cell stimulating expression TLR2 with candidate inhibitor.Then, stimulate with pro-inflammatory cytokine.Stimulating method can use and well known to a person skilled in the art any means.As stimulating method, the method etc. as co-cultured with candidate inhibitor or pro-inflammatory cytokine by cell can be enumerated, but be not limited to this.After stimulating with pro-inflammatory cytokine, measure the expression of the struvite immunity-associated cell factor in described cell.The assay method of the expression of the struvite immunity-associated cell factor is not particularly limited, it is possible to use well known to a person skilled in the art all methods.The most i.e. can measure the expression of mRNA, it is also possible to measure the protein content of secretion.Measuring method can change according to the index of the struvite immunity-associated cell factor expression amount measured.As the example, in the case of measuring RNA amount, real-time quantitative PCR method can be enumerated, in the case of measuring secretory protein, absorption photometry etc. can be enumerated, but be not limited to this.
Stimulation based on pro-inflammatory cytokine, from the standpoint of eliminating candidate inhibitor acts on the impact that pro-inflammatory cytokine itself produces, is preferably carried out under the conditions of without candidate inhibitor.Thus, it is preferable between (1) operation and (2) operation, farther include the operation of the cell after cleaning (1) moderate stimulation.This way it is possible to avoid the impact that pro-inflammatory cytokine itself is caused by candidate inhibitor, the struvite immunization inhibitor directly acting on TLR express cell can be screened more accurately.
As it has been described above, the cell in the present invention can use arbitrary cell, but from screening from the standpoint of enteral plays the struvite immunization inhibitor of effect, intestinal epithelial cell from pig or people is preferably used.
As pro-inflammatory cytokine, as long as struvite immunity can be induced, it is possible to use any pro-inflammatory cytokine well known by persons skilled in the art.Such as there are the thalline such as compound, escherichia coli etc. such as lipopolysaccharide, viral dsRNA, polyinosinic acid (PolyI:C) (synthesis dsRNA), but are not limited to this.Consider from inducing struvite immunology at enteral, preferably enterotoxigenic E．Coli (ETEC).
As it has been described above, express the cell of TLR2, both can be to convert cell, it is also possible to be to separate cell, but if it is considered that intracellular protein expression profile, then the cell of natural expression TLR be preferably used.Further, as an object of the present invention, biological in order to find excellent immunity, the intestinal cell such as intestinal epithelial cell, Caco2 cell, most preferably with normal intestinal epithelial cell are preferably used.
Measure the struvite immunity-associated cell factor of expression, as long as the cytokine relevant to inflammatory reaction, can be measured any one, it is also possible to screen one or more preferred struvite immunity-associated cell factors and measure.As the cytokine relevant to inflammatory reaction used in the present invention, such as IL-1 α, IL-1 β, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, IFN-α, IFN-β, IFN-γ, MCP-1, TNF-α, TGF-β, LIF etc. can be enumerated, but be not limited to this.Present inventor has performed further investigation, result has filtered out the three kinds of cytokines (IL-6, IL-8 and MCP-1) induced the most by force from the 12 kinds of cytokines (IL-1 α, IL-1 β, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, MCP-1, TNF-α, TGF-β, LIF) carrying out testing intestinal epithelial cell by enterotoxigenic E．Coli.Thus, struvite the immunity-associated cell factor preferred IL-6, IL-8 and MCP-1 tri-kinds of measurement.
Above-mentioned screening such as can be carried out by following method, but is not limited to the method.
BIE cell is carried out inoculated and cultured according to the method identical with following embodiment 1, with ETEC, it is stimulated after 3 days.After ETEC stimulates 12 hours, use real-time PCR methodology that 12 kinds of cytokines (IL-1 α, IL-1 β, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, MCP-1, TNF-α, TGF-β, LIF) are measured the expression of mRNA.By this result compared with the comparison that stimulation is not carried out, the expression of IL-1 α, IL-6, IL-8 and MCP-1 significantly increases, therefore using these as the alternate cell factor.Then, BIE cell carries out continuing to cultivate after inoculating 3 further, is 2 as the pre-stimulation scheduled time, then stimulates with ETEC.After ETEC stimulates 12 hours, use the real-time PCR methodology expression to described alternate cell factor determination mRNA.By result compared with the comparison that stimulation is not carried out, the expression of IL-6, IL-8 and MCP-1 significantly increases, the struvite immunity-associated cell factors (with reference to Fig. 4) being therefore measurement by these three kinds screenings.
As struvite immunization inhibitor, due to the struvite immunization in its suppression intestinal epithelial cell, the struvite immunization inhibitor the most preferably can being orally ingested.Further, the biological or derivatives thereof (immunogenicity microorganism) of struvite immunization inhibitor more preferably immunity.
In the present invention, " immunogenicity microorganism " refer to come from the immunoregulatory factor that immunity is biological.As the material being included in immunogenicity microorganism, can enumerate as constituted the Peptidoglycan of cell wall, teichoic acid, lipoteichoic acid and thalline exo polysaccharides (EPS) as thalline surface layer composition；Further, as composition in thalline, the nucleic acid compositions such as chromosomal DNA etc. can be enumerated, as the few DNA of its Rastriction enzyme fragment or include the cytoplasmic components of various pherons etc., but be not limited to this.
According to the screening technique of the present invention, excellent struvite immunization inhibitor can be filtered out.Thus in the present invention, also including the struvite immunization inhibitor that the method by the present invention filters out, the most struvite immunization inhibition immunity is biological.Biological as this immunity, from the viewpoint of broadly belonging to probiotic bacteria, preferably Lactobacillus bacterium or Bifidobacterium bacterium.
The present inventor is according to the screening technique of the present invention, the immunity biology with the struvite immunization inhibition comprised in the present invention has been isolated from numerous Lactobacillus bacterium, wherein biological as gratifying immunity, isolate OLL2768 bacterial strain and TL2937 bacterial strain.
The Lactobacillus bacterium of inclusion in the present invention, there is following feature, by itself and intestinal epithelial cell are co-cultured more than 2 days, the expression compared with the above-mentioned struvite immunity correlation factor in the intestinal epithelial cell not co-cultured of one or more the struvite immunity-associated cell factors in the intestinal epithelial cell that suppression is exposed in pro-inflammatory cytokine.Lactobacillus bacterium is one of representative genus of lactic acid bacteria, is Gram-positive bacillus, according to the difference planted, including only producing lactic acid (homofermentative lactic) and producing (heterolactic fermentation) type of product beyond lactic acid.
The struvite immunity-associated cell factor of suppression, as long as become the cytokine of inflammatory reaction reason, but the struvite immunity-associated cell factor preferably significantly increased by the stimulation expression of pro-inflammatory cytokine.In BIE cell, from the standpoint of being significantly increased by the stimulation expression of enterotoxigenic E．Coli, preferably any one in IL-6, IL-8 or MCP-1, more preferably suppresses the two kinds of factors selected from IL-6, IL-8 and MCP-1, most preferably suppresses tri-kinds of factors of IL-6, IL-8 and MCP-1.
In an embodiment of the invention, the expression of Lactobacillus bacterium suppression 30% the above struvite immunity-associated cell factor of the present invention.
nullAs the Lactobacillus bacterium comprised in the present invention，，Can enumerate such as lactobacillus delbruockii subspecies bulgaricus (Lactobacillusdelbrueckiisubsp.Burgaricus)、Lactobacillus delbrueckii subsp. lactis (Lactobacillusdelbrueckiisubsp.Lactis)、Lactobacillus casei (Lactobacilluscasei)、Lactobacillus helveticus (Lactobacillushelveticus)、Bacillus acidophilus (Lactobacillusacidophilus)、Lactobacillus crispatus (Lactobacilluscrispatus)、Food starch milk bacillus (Lactobacillusamylovorus) (hereinafter referred to as L.amylovorus)、Chicken lactobacillus (Lactobacillusgallinarum)、Lactobacillus gasseri (Lactobacillusgasseri) (hereinafter referred to as L.gasseri)、Lactobacillus oris (Lactobacillusoris)、Lactobacillus rhamnosus (Lactobacillusrhamnosus)、Lactobacillus johnsonii (Lactobacillusjohnsonii)、Lactobacillus fermenti (Lactobacillusfermentum)、Lactobacillus brevis (Lactobacillusbrevis)、Lactobacillus plantarum (Lactobacillusplantarum) etc.，But it is not limited to this.Preferably lactobacillus casei (Lactobacilluscasei), sieve Yi Shi lactobacillus (Lactobacillusreuteri), lactobacillus rhamnosus (Lactobacillusrhamnosus), Lactobacillus salivarius (Lactobacillussalivarius), Lactobacillus Jensenii (Lactobacillusjensenii) or Lactobacillus gasseri (Lactobacillusgasseri) etc..
The OLL2768 strain of the present invention has carried out preservation on March 17th, 2010 in Independent Administrative Leged Industrial Technology Complex Inst (a kind of ground 1 central authorities the 6th of east, Zhu Bo city, Ibaraki, Japan 1 fourth mesh), preserving number is: FERMBP-11244, and described OLL2768 strain is the lactobacillus casei with following characteristics.
(a) morphological properties
Bacillus
(b) cultural property
Culture medium title: lactobacillus MRS culture medium (trypsin Difco), Ref.No.288130)
PH value: without adjusting
Cultivation temperature: 37 DEG C
Incubation time: 18 hours
(1) shape: circular
(2) diameter: 1 ~ 2mm
(3) tone: white
(4) protuberance state: hemispherical
(5) edge: Quan Yuan
(6) surface configuration: smooth
(7) transparency: opaque
(8) viscosity: butter shape
(c) physiological characteristics
(1) Gram’s staining: positive
(2) lactate fermentation form: homofermentative lactic
(3) aerobism: amphimicrobian
(4) developmental temperature: 15 DEG C+, 45 DEG C-
The TL2937 strain of the present invention has carried out preservation on May 14th, 2010 in Independent Administrative Leged Industrial Technology Complex Inst (a kind of ground 1 central authorities the 6th of east, Zhu Bo city, Ibaraki, Japan 1 fourth mesh), preserving number is: FERMBP11272, and it is the Lactobacillus Jensenii with following characteristics.
(a) morphological properties
Bacillus
(b) cultural property
Culture medium title: lactobacillus MRS culture medium (trypsin, Ref.No.288130)
PH value: without adjusting
Cultivation temperature: 37 DEG C
Incubation time: 18 hours
(1) shape: circular
(2) diameter: 1 ~ 2mm
(3) tone: white
(4) protuberance state: semi-transparent specular
(5) edge: Quan Yuan
(6) surface profile: smooth
(7) transparency: opaque
(8) viscosity: butter shape
(c) physiological characteristics
(1) Gram’s staining: positive
(2) lactate fermentation form: homofermentative lactic lactic acid optical activity: D type
(3) aerobism: amphimicrobian
(4) developmental temperature: 15 DEG C-, 45 DEG C+
Bacterium or the immunity of the present invention are biological, can suppress the struvite immunization of intestinal epithelial cell, therefore comprise the inflammation inhibitor compositions of at least one in described bacterium or immunity biology and fall within the present invention.This inflammation inhibitor compositions, due to biological containing immunity, by oral administration, can improve the enteral environment being administered object, and its result can prevent and/or dispose enteral inflammatory disease.Thus it is preferred that the inflammation inhibitor compositions of the present invention is inflammation inhibitor compositions for the purpose.
The inflammation inhibitor compositions of the present invention, owing to can pass through oral administration, therefore can make it be included in food compositions.This food compositions is a kind of effect by contained inflammation inhibitor compositions, can improve the food compositions of the individual enteral environment of picked-up.As the form of food compositions, as long as the survivable form of bacterium, without particular determination.As the obtainable form of this food compositions, such as the milk product such as Yoghourt, cheese, powder or tablet, animal feed etc. can be enumerated, but be not limited to this.
Thus, the inflammation inhibitor compositions of the present invention, it can be made to be included in animal feed compositions.This fodder compound can also improve the enteral environment that picked-up is individual.The fodder compound of the present invention, can serve as the feedstuff with any one animal in the animal of intestinal flora, can enumerate as pet animals such as dog, cat, Mus, Cavia porcelluss, or for including the domestic animal of cattle, horse, pig, chicken etc..
Hereinafter, according to embodiment, the present invention will be described, but embodiment is the example of the present invention, and the present invention is not limited to this.
Embodiment
Example 1: the screening that anti-inflammatory immunity based on BIE cell is biological
Use gut epithelium (BIE) cell of calf, use enterotoxigenic E.Coli E.coli(ETEC987P strain as pro-inflammatory cytokine), 18 strains of lactic acid bacteria described in table 1 below are studied.Heat sterilization process in 30 minutes is carried out by its each comfortable 56 DEG C.
Table 1 test strain table
| Bacterial strain | Kind |
| MEP221101 | Sieve Yi Shi lactobacillus |
| MEP221102 | Sieve Yi Shi lactobacillus |
| MEP221103 | Lactobacillus casei |
| OLL2768 | Lactobacillus casei |
| MEP221104 | Lactobacillus casei |
| MEP221105 | Lactobacillus casei |
| MEP221106 | Lactobacillus casei |
| MEP221107 | Lactobacillus casei |
| MEP221108 | Lactobacillus casei |
| MEP221109 | Lactobacillus casei |
| MEP221110 | Lactobacillus rhamnosus |
| MEP221111 | Lactobacillus rhamnosus |
| MEP221112 | Lactobacillus rhamnosus |
| MEP221113 | Lactobacillus rhamnosus |
| MEP221114 | Lactobacillus casei |
| MEP221115 | Lactobacillus casei |
| TL2937 | Lactobacillus Jensenii |
| MEP221117 | Lactobacillus gasseri |
The preparation of (a) lactic acid bacteria
With MRS culture medium (trypsin, Detroit, the U.S.), bacterial strain carried out (37 DEG C, 16h) after 3 successive transfer culture, fully clean with PBS.30 minutes heat sterilizations are carried out in 56 DEG C after cleaning.Again with PBS twice, then at DMEM(10%FCS, 1% streptomycin/penicillin) in settling flux.
(b) BIE cytositimulation
With 1.5 × 10 in 24 orifice plates4Individual/hole inoculation BIE cell, cultivates 3.Cultivate the 3rd and stimulate 2 for examination lactic acid bacteria (100MOI).After stimulation, with 3 plate holes of PBS, with 5 × 107The ETEC of individual/ml stimulates.
C () is by BIE cell extraction total serum IgE (TotalRNA) and synthesis cDNA
After removing culture medium and cleaning, by using the test kit of TRIzol reagent to extract total serum IgE.QuantiTect Reverse Transcriptase kit (test kit (Qiagen)) is used to be synthesized cDNA by total serum IgE.
D () is quantitative to cattle cytokines mRNA by real-time PCR methodology
The cDNA obtained is used to carry out real-time RT-PCR.PCR reaction solution is by employing PlatinumTMSYBRTMGreenqPCRSuperMix-UDGwithROX(hero company (Invitrogen)) system implement, by using 7300 real-time PCR systems (Applied Biosystems, Inc., Lincoln, CDF, the U.S.) to be analyzed and resolve.By the amplification curve group obtained, by any fluorescent value Ct(threshold cycle (Thresholdcycle) in the amplification region of exponential function) it is calculated standard curve.Calculated the mrna expression amount of various cytokines in each sample by this standard curve, by this value divided by the mrna expression amount of the beta-actin in each sample, compareing with ETEC is that relative expression intensities when 1 compares.With Student ' st inspection resolve relative to ETEC comparison 2 groups between statistical significant difference.
Result is as shown in Figure 1.From three the cytokine indexs stimulating induction because of ETEC, having filtered out OLL2768 strain, it especially suppresses IL-6 and IL-8 of strongly expressed, and suppression MCP-1 expresses.
Example 2: biological based on the immunity in BIE cell, the expression of the signal inhibitive factor of Toll-like receptor
With 1.5 × 10 in 24 orifice plates4Individual/hole inoculation BIE cell, cultivates 3.Cultivate the 3rd with chemosynthesis triacylate lipopeptid Pam3CSK4(200ng/ml) or OLL2768 strain (5 × 107nullIndividual/1ml) stimulate 12、24、36、48 hours，Toll-like receptor signal inhibitive factor (MKP-1(MAPKphosphatase1:MAPK phosphatase 1) is resolved by Real-time PCR assay、IRAK-M(Interleukin-1Recepter-AssociatedKinaseM: interleukin 1 receptor associated kinase M)、SIGIRR(SingleIgIL-1-RelatedRecepter: single IgIL-1 associated receptor)、BCL-3(B-cellCLL/Lymphoma3:B cell CLL/ lymphoma 3)、Tollip(Toll-InteractingProtein:Toll action protein)、A20(TNFAIP3((TumorNecrosisFactor,Alpha-InducedProtein3: tumor necrosis factor α induced protein 3)) expression.
It addition, after stimulating BIE cell 48 hours with OLL2768 strain, with three plate holes of PBS, to 5 × 107The expression of inhibitive factor after the ETEC of individual/ml stimulates 3,6,12 hours has been also carried out resolving.
Use and example 1(c from BIE cell extraction total serum IgE and synthesis cDNA) identical method implements, uses the mensuration of cattle cytokines mRNA based on real-time quantitative RT-PCR method and example 1(d) identical assay method.
Result is as shown in Figures 2 and 3.Fig. 2 is the chart of the expression representing each cytokine based on OLL2768 strain stimulation, and its display is by stimulating BIE cell 48 hours with OLL2768 strain, and the expression of Tollip and BCL3 strengthens.It addition, Fig. 3 is to represent to stimulate after 48 hours with OLL2768 strain, give the chart of the change of each cytokine-expressing amount when ETEC stimulates further.Its display is by stimulating 6 hours with ETEC further, and the expression of Tollip strengthens.And can confirm that the expression of BCL3 also has the tendency of rising.
By these as a result, it is possible to filter out the candidate bacterial strain (OLL2768 strain) biological as immunity, inflammatory response based on ETEC when it can alleviate use BIE cell.And can speculate that its activity expression structure is that expression based on Toll-like receptor signal inhibitive factor strengthens, suppress NF-kB transcriptional activity.
Example 3: the anti-inflammatory activity rating in pig gut epithelium (PIE) cell
Use PIE cell, use method same as Example 1 to evaluate anti-inflammatory activity.That is, with 3 × 10 in 12 orifice plates4Individual/hole inoculation PIE cell, at 37 DEG C, 5%CO2Under the conditions of cultivate 3 days.500 μ l are added respectively for examination lactic acid bacteria, at 37 DEG C, 5%CO with each in 100MOI hole beyond culture medium comparison and ETEC comparison2Under the conditions of cultivate 48 hours.With PBS 12 orifice plate, after adding the DMEM culture medium (10%FCS, 1%SP) of 500 μ l in each hole, the hole beyond culture medium check plot is inoculated ETEC(987P strain) dead thalline is to 5.0 × 107Individual/hole (100MOI), stimulates PIE cell 12 hours.Stimulate the TRIzol adding 500 μ l in backward each hole, reclaim cytolysate by pipet.Extract total serum IgE, calculated the mrna expression amount of IL-6, IL-8 and MCP-1 in each reagent by real-time PCR methodology, by this value divided by the mrna expression amount of the beta-actin in each sample, be that relative expression quantity when 100 is evaluated with ETEC comparison.With Student ' st inspection resolve relative to ETEC comparison 2 groups between statistical significant difference.
In these 7 kinds of bacterial strains of OLL2768, TL2937, MEP221102, MEP221104, MEP221111, MEP221113, MEP221115, it was observed that the significance suppression of IL-6.And observed these bacterial strains in the expression of MCP-1, also have a certain degree of inhibitory activity, expressing of IL-8 has low tendency with other compared with examination lactic acid bacterias.Wherein, in TL-2937 strain, relative to positive control, it was observed that the IL-6 inhibitory activity of more than 60%, consider the result of example 1, it is believed that it is the biological candidate bacterial strain of the immunity that other are useful simultaneously.
Example 4: the antiviral property inflammatory activity in swine intestinal epithelium cells is evaluated
The preparation of (a) PIE cell and stimulation
In 24 orifice plates scribble NTx protein coating, 500 μ l concentration are added in every hole is 1 × 104The cell of individual/ml, at 37 DEG C, 5%CO2Under the conditions of cultivate 3 days.After carrying out the culture medium replacing of PIE cell, stimulate the biological candidate bacterial strain of the immunity shown in table 1 with each 100MOI, at 37 DEG C, 5%CO2Under the conditions of cultivate 0 ~ 48 hour.After pre-stimulation based on lactic acid bacteria, with PBS three times, do not remain in confirming hole with optical microscope for examination lactic acid bacteria.Then in the DMEM being added with 10%FCS, 1% Pen .-Strep, polyinosinic acid is added, by its concentration dilution to 12.5 μ g/ml being added with the DMEM(of polyinosinic acid (polyI:C)) replace the culture medium in each hole, cultivate 12 hours.The hole setting non-stimulated as comparison and the hole only stimulated with polyinosinic acid.
(b): antiviral property and the evaluation of anti-inflammatory activity
From the PIE cell cultivated, extract mRNA, synthesize cDNA.With example 1(d) identical, by real-time PCR methodology, the parsing IFN-β as the interferon type Ⅰ of antiviral property index, the induced expression of the MCP-1 as anti-inflammatory index.That is, using cDNA as template, the primer of use carries out expressing parsing.The expression of cytokine gene resolves, and is carried out by ABIPRISM7300 real-time PCR system (Applied Biosystems, Inc.).Standard curve is from the curve obtained as gradient dilution plasmid the result that is analyzed, by the Ct(threshold cycle of any fluorescent value in the amplification region of exponential function) calculated standard straight-line.By the Ct in each sample and standard straight-line, the mrna expression amount of cytokine in separate sample under the conditions of calculating respectively.Obtain the expression of the beta-actin of same sample, by the value standardization of each expression, compare polyinosinic acid (Poly(I:C)) it is relative expression intensities when 100.Resolve relative to polyinosinic acid (Poly(I:C) with Student ' st inspection) statistical significant difference between compare 2 groups.
When PIE cell is carried out polyinosinic acid stimulation, in IFN-β and MCP-1, it is observed that the expression of process in time changes on gene level.Especially, can confirm that substantially reducing of stimulating that IFN-β after 12 hours expresses and the expression of MCP-1 increases, therefore using both cytokines as immune parameter, after being stimulated 12 hours by polyinosinic acid, it is determined as optimal evaluation time.Use this appraisement system, during candidate bacterial strain pre-stimulation PIE cell biological with immunity, by the pre-stimulation of 12 hours, the bacterial strain expressing enhancing as induction IFN-β, can obtain MEP221106, MEP221108, be stimulated by 48 hours, TL2937 shows stronger trend (Fig. 5).nullAdditionally，By the pre-stimulation of 12 hours，At MEP221101、MEP221102、MEP221114、MEP221115、In five kinds of bacterial strains of MEP221117，And stimulated by 24 hours，At MEP221101、MEP221102、MEP221103、OLL2768、MEP221104、MEP221105、MEP221106、MEP221108、MEP221109、MEP221110、MEP221111、MEP221112、MEP221114、MEP221115、TL2937、16 kinds of bacterial strains of MEP221117 confirm the significance expression inhibiting (Fig. 6) of MCP-1.From result above, by using the in-vitro evaluation system of PIE cell, can expect for index screening and to evaluate immunity biology with the enhancing of antiviral property cytokine and the suppression of viral inflammation.
Example 5: for the effect of antibacterial in the feedstuff of productivity ratio or immunocompetence
Totally 20 pigs (female) for examination, import with LWD system when 2 week old, are divided into 5,4 every district of district, give commercially available milk replacer and raise and train to 3 week old.The later feedstuff of 4 week old be containing be suitable for the stage of growth from breast feedstuff, four kinds of feedstuffs that basic ingredient is different, to add the feedstuff of antibacterial for 100%, the feedstuff 0% phase mixing identical with the basic ingredient without antibacterial, thus set the feedstuff of 50% input antibacterial and four districts of totally four feedstuff conditions of 25%, make its be arbitrarily satiated with food (table 2 and table 3) respectively.
Normal feedstuff composition beyond table 2 antibacterial
TDN: digestible nutrient total amount
Table 3 trial zone and mixed proportion
| District | Head number | Add antibacterial (%) | Without antibacterial (%) |
| 1 | 5 | 0 | 100 |
| 2 | 5 | 25 | 75 |
| 3 | 5 | 50 | 50 |
| 4 | 5 | 100 | 0 |
Carrying out measured body weight every two weeks, gather nasal mucus and blood sampling, the blood gathered from part is rapidly separated blood plasma, with nasal mucus the most all at-80 DEG C freezen protective to when analyzing.Blood plasma is used for measuring Metabolite (gross protein, glucose, T-CHOL, nitrate nitrogen, triacylglycerol (triacylglycerol), calcium) and CRP matter (CPR) concentration, evaluates the total white blood cell count after just gathering in whole blood, granulocyte number ratio, macrophage activity, antibody-producing ability and complement the 2nd pathway activity to lymphocyte number respectively.Further, fresh feces is gathered from each district.Use nasal mucus to carry out mycoplasma infection inspection respectively, use feces to carry out the infection inspection of pathogenic Escherichia coli (k88 and k99).Body weight is delivered for sale when reaching 115kg, massacres after dismembering, investigates carcass achievement.
Result is as shown in Fig. 7 ~ 9 and table 4.Understand with whether to add antibacterial unrelated, more weak to the infection of pathogenic Escherichia coli to 8 week old, during 13 week old to 15 week old, there is strong infectivity.After 17 week old, the infection that antibacterial adding proportion is high is weak, and (Fig. 7) is infected by force in low generation.After 13 week old, the 4th district of 100% antibacterial, CRP concentration within normal range (Fig. 8) in blood plasma.In terms of Metabolite concentration, do not find between week old and interval, there is big difference.During immunocompetence after 13 week old is evaluated, especially in the granulocyte number ratio to lymphocyte number, the district that with the addition of antibacterial significantly reduces (P ＜ 0.05) (Fig. 9).Can confirm that the mycoplasma infection in nasal mucus, unrelated with whether adding antibacterial.Even for arbitrary carcass achievement, in terms of the effect delivering age in days for sale, do not see significance yet.When delivering for sale on body weight and carcass weight, in interval unknown significance difference, on the other hand, backfat thickness is at the side that with the addition of antibacterial the thinnest (P ＜ 0.05) (table 4).
Body weight, carcass weight and the meansigma methods of backfat thickness when table 4 is delivered for sale
A, b: there is between distinct symbols significant difference (P ＜ 0.05)
Example 6: inflammation inhibitor is for productivity ratio or the effect of immunocompetence
Totally 40 for the pig (female or castrating male) of examination, imports with LWD system when 3 week old, is divided into 5,8 every district of district, gives commercially available milk replacer and raise and train to 4 week old.The later feedstuff of 36 ages in days be containing be suitable for the stage of growth from breast feedstuff, the Three feed (table 1) that basic ingredient is different, prepare respectively 3 kind 100% add the feedstuff of antibacterial, feedstuff that 3 kind of 0% basic ingredient without antibacterial is identical, six kinds of feedstuffs make it arbitrarily be satiated with food altogether.Further, from 3 week old to 15 week old, as the substitute of antibacterial, give 2 kinds of biological lactic acid bacterias (L1: Lactobacillus Jensenii TL2937, L2: Lactobacillus plantarum MEP221118) (table 5) of immunity with other containers and culture fluid simultaneously.Bacterium number in culture fluid is 3 × 108cfu/g.Administered dose is 3g/ body weight kg every day every.Culture fluid is with the milk surum of enzyme resolution process.Mode is given as shown in table 5 to 8 districts.
Sample collection is the most same as Example 5.Research project is removing antibody-producing ability evaluation from example 5, and additionally the kind apoplexy due to endogenous wind at the pathogenic Escherichia coli of detection increases ETEC987P.Deliver for sale and the investigation of carcass achievement uses method same as Example 5.
Table 5 trial zone gives with feedstuff
| District | Head number | Add antibacterial (%) | Without antibacterial (%) | Antibacterial sub |
| 1 | 5 | × | ○ | Without adding |
| 2 | 5 | × | ○ | It is only culture medium |
| 3 | 5 | × | ○ | L1 |
| 4 | 5 | × | ○ | L2 |
| 5 | 5 | ○ | × | Without adding |
| 6 | 5 | ○ | × | It is only culture medium |
| 7 | 5 | ○ | × | L1 |
| 8 | 5 | ○ | × | L2 |
Result is as shown in Figure 10 ~ 14, during 4 week old to 8 week old, unrelated with whether adding antibacterial, although to have power, but can confirm that the infection to pathogenic Escherichia coli.Especially giving district at the L1 added without antibacterial, between 13 week old to 15 week old, the infection to pathogenic Escherichia coli disappears.On the other hand, add district at antibacterial, although can confirm that power, but can confirm that to 15 week old and infect (Figure 10).CRP concentration in blood plasma, from 13 week old to 15 week old, compared with the check plot added without thalline, the L1 added without antibacterial give district within normal range the lowest (P ＜ 0.05), even if additionally adding antibacterial, L1 gives district low trend (Figure 11) within normal range.As immunocompetence evaluation, it is notable the highest (P ＜ 0.05) (Figure 12) that complement the 2nd pathway activity of especially 13 week old to 15 week old gives district at the L1 added without antibacterial.About body weight, giving district at the L1 added without antibacterial, weightening finish is notable optimal, 115kg(P ＜ 0.05 is the most averagely reached during 22 week old), it addition, during with week old, L1, L2 and culture medium give district, with without compared with giving district, have the trend (Figure 13) of increase.On the other hand, carcass weight at antibacterial without adding in district, antibacterial be each showed no significant difference in adding district, both compare, and antibacterial adds district and has the least trend (Figure 14).
Knowable to the result of example 5 and example 6, maintaining mother to pass the effect of antibody before 8 week old, the infection to pathogenic Escherichia coli is weak.But, have strong infection, the trend with step-down that then antibacterial adding proportion is high from 13 week old to 15 week old are unrelated with whether adding antibacterial, antibacterial period to the effect of pathogenic Escherichia coli can not be expected thus it is speculated that exist.Additionally antibacterial adding proportion height person, in blood plasma, CRP concentration is low, owing to it is in the range of normal, it may thus be appreciated that it significantly inhibits inflammatory response.Further, by adding antibacterial, the ratio of lymphocyte number is reduced by granulocyte number, therefore by the effect of antibacterial, can suppress the entrance of pathogenic Escherichia coli, and natural immune system response is maintained at normal condition by result.
Giving experimental result and understand from the biological lactic acid bacteria of immunity, and gives unrelated, is or strong or weak Infection Status, but is giving without the L1 in the environment of antibacterial, the pathogenic Escherichia coli infection high inhibition to 13 week old to 15 week old before 8 week old.It addition, by giving the L1 added without antibacterial, the CRP concentration in blood plasma is the lowest, suppression is within normal range, the notable rising of complement the 2nd pathway activity, therefore L1 suppression pathogenic Escherichia coli infects, and can expect immune activation and the effect of suppression inflammatory response.Further, the L1 added without antibacterial gives the body weight in district, has reached the 115kg being suitable for delivering for sale when 22 week old, within 2 weeks ahead of time, has completed relative to standard.It addition, carcass weight zero difference compared with other districts, therefore productivity ratio obtains improving.
In summary, as the substitute of the antibacterial maintaining productivity ratio, the biological lactic acid bacteria L1 of immunity not only suppresses pathogenic Escherichia coli to infect, and can expect Immunestimulatory effect, and improve the productivity ratio delivered for sale in early days based on raising body weight and be made that contribution.
Industrial applicibility
Pass through the present invention, the immunity that can screen the excellent benefit that can suppress bowl inflammatory immunne response is biological, and, the bacterial strain OLL2768 strain screened by the method for the present invention and TL2937 strain, the immunity being excellent is biological, can expect the application as antibacterial succedaneum.
Claims (4)
1. preserving number be FERMBP-11272 Lactobacillus Jensenii (Lactobacillusjensenii) application in preparing inflammation inhibitor compositions of the TL2937 bacterial strain.
Apply the most according to claim 1, it is characterised in that described inflammation inhibitor compositions is used for preventing and/or dispose inflammatory disease in intestinal.
3. preserving number be FERMBP-11272 Lactobacillus Jensenii (Lactobacillusjensenii) application in the food compositions of preparation suppression inflammation of the TL2937 bacterial strain.
4. preserving number be FERMBP-11272 Lactobacillus Jensenii (Lactobacillusjensenii) application in the fodder compound of preparation suppression inflammation of the TL2937 bacterial strain.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| PCT/JP2010/066357 WO2011118060A1 (en) | 2010-03-26 | 2010-09-22 | Method for screening intestinal immunity suppression agents |
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| CN103087963B (en) * | 2013-01-31 | 2015-03-04 | 武汉工业学院 | Method for screening probiotics |
| JP5927730B2 (en) * | 2013-04-11 | 2016-06-01 | アサヒグループホールディングス株式会社 | Screening method for lactic acid bacteria having immunomodulating action |
| CN103289927B (en) * | 2013-05-30 | 2015-03-25 | 中国农业科学院哈尔滨兽医研究所 | Lactobacillus salivarius strain and application thereof |
| CN105861717A (en) * | 2016-05-27 | 2016-08-17 | 苏州铭冠软件科技有限公司 | Detection method for improved variety breeding |
| WO2018181619A1 (en) * | 2017-03-28 | 2018-10-04 | 味の素株式会社 | Food for improving intraintestinal environment |
| TWI707689B (en) * | 2019-06-11 | 2020-10-21 | 大江生醫股份有限公司 | Immunomodulatory probiotic bacteria and uses thereof |
| CN112190600B (en) * | 2020-09-11 | 2022-04-26 | 杭州娃哈哈科技有限公司 | Application of lactobacillus plantarum in preparation of composition for relieving chronic inflammation of organism |
| CN114908009B (en) * | 2022-05-10 | 2023-06-23 | 新疆农业大学 | Lactobacillus mucilaginosus PR63 and application thereof |
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| PE20030283A1 (en) * | 2001-07-26 | 2003-05-01 | Alimentary Health Ltd | LACTOBACILLUS CASEI STRAINS |
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Non-Patent Citations (3)
| Title |
|---|
| Development of molecular immunoassay system for probiotics via toll-like receptors bases on food immunology;KITAZAWA H.等;《Anim Sci J》;20081231;第79卷(第1期);摘要，图6 * |
| Toll-like receptor 4 and cytokine expression involved in functional immune response in an originally established porcine intestinal epitheliocyte cell line;MOUE M.等;《BBA-General Subjects》;20081231;第1780卷(第2期);134-144 * |
| 免疫生物在肠道上皮细胞中的抗炎作用;岛津等;《日本畜产学会第110次大会演讲摘要》;20081231;X27-05 * |
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| CN102884174A (en) | 2013-01-16 |
| JPWO2011118060A1 (en) | 2013-07-04 |
| WO2011118060A1 (en) | 2011-09-29 |
| HK1176379A1 (en) | 2013-07-26 |
| JP5852558B2 (en) | 2016-02-03 |
| JP2016047057A (en) | 2016-04-07 |
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